Glucose, fructose, mannose and/or glucose‐1‐phosphatereleasing activity stains for glycosidases and glycosyltransferases in gels after isoelectric focusing

Abstract

beta-Fructofuranosidase, alpha-glucosidase, beta-glucosidase, alpha-mannosidase, beta-mannosidase, sucrose phosphorylase, glucosyltransferase and fructosyltransferase were separated by isoelectric focusing and sensitively detected to be slightly diffuse and insoluble spots in thin-layer gels, supported by a glass plate, by release of monosugars or a sugar phosphate, followed by conversion to glucose-6-phosphate (G6P) and then by reduction of NADP+ to NADPH, terminated by the formation of reduced Nitroblue Tetrazolium (NBT). Approximately 1-10 mU of enzyme was focused and the gel, after washing with a buffer, was partially dried and directly stained by uniformly spreading on the gel surface a staining medium containing sucrose or nitrophenyl glycosides as substrates, intermediary enzymes such as hexokinase, mutase and/or isomerase, NADP+, ATP, Mg+, phenazine methosulfate (PMS) and NBT. Specific staining procedures for each of these activities, on sucrose or on the glycosides as substrates, and staining procedures for multiple activities are described, with the conditions necessary for optimal development.

Renaturation and activity staining of glycosidases and glycosyltransferases in gels after sodium dodecyl sulfate‐electrophoresis

“…The staining system via glucose-6-phosphate for sugarreleasing activities after IEF has been previously reported in details [7] and is also in this report shown to be highly sensitive after SDS-PAGE as well as 2-DE. The amounts of the five renaturable enzymes loaded in this study, including 2-DE, were 2 m u -10 mU or 2 pL and almost the same amounts were loaded on IEF [7], indicating that most of them were well renatured with regard to activity, except the clMase with a half or a third of renaturation.…”

“…The amounts of the five renaturable enzymes loaded in this study, including 2-DE, were 2 m u -10 mU or 2 pL and almost the same amounts were loaded on IEF [7], indicating that most of them were well renatured with regard to activity, except the clMase with a half or a third of renaturation. It should also be emphasized that trace amounts of SDS probably remaining in the gel, neutralized with an excess amount of Triton X-100, never exhibited an inhibition on components of the staining system, including intermediary enzymes, coenzymes and dyes.…”

“…Renaturation of enzymes after high-resolution separation by SDS-PAGE or 2-DE, followed by sensitive activity stains in gels, affords the analytical advantage of direct identification of each specific enzyme in crude cell extracts or culture supernatants. This study demonstrates the renaturability of eight SDS-denatured glycosidases and glycosyltransferases by applying the sensitive staining system for sugar-releasing activities which we recently reported [7], and also by applying the one-and two-dimensional electrophoresis systems that are significantly improved on our previous systems [6], using a single thin-layer gel covalently bound to a glass plate [8]. …”

“…The gel was then washed with 0.4% Triton X-100, uniformly spread with the Ampholine mixture and separated by isoelectric focusing in the second dimension, and finally stained with the media for the sugar-releasing activities. PFase (M, 270 000, p l 4.1), aMase (MI 190 000, p16.2) and SPase (M, 56 000, pl5.0) [7] were detected in the layer gel at the positions expected by their M, and p l values (Fig. 3A).…”